Jel 42 is an IgG which binds to the small bacterial protein, HPr and the structure of the complex is known at high resolution. The IgG was expressed as a single chain variable fragment (scFv) and the binding to HPr was assessed by fluorescence polarization of fluorescein-labelled HPr. The binding constant for the IgG was about 20-fold higher than the scFv. Inspection of the structure of the complex suggested that it might be possible to convert the scFv into a bond-specific protease by the introduction of three catalytic residues: a glutamate to increase the nucleophilicity of a nearby water molecule, a lysine to increase the polarizability of the carbonyl group and a histidine to provide a proton to convert the amine into a better leaving group. By trial and error it was found that a fourth residue had to be converted into glycine in order to maintain the integrity of complimentarity-determining region three of the heavy chain (CDRH3) at the binding interface. The resulting quadruple mutant still bound to HPr and unlike other mutants, showed weak protease activity as judged from the fluorescence polarization assay. The activity was maximum at pH 6 consistent with a requirement for a protonated histidine residue. With the aid of HPr fluorescein-labelled at two different positions, it was demonstrated that the size of the products was consistent with cleavage occurring in the vicinity of the target peptide bond. The activity was specific for HPr since an excess of bovine serum albumin did not interfere with the reaction.